NAP-2 polypeptides and methods for modulating immune system activity in heart tissue

ABSTRACT

This document provides methods and materials for reducing the risk of major adverse cardiac events. For example, methods and materials for identifying patients at risk of experiencing a major adverse cardiac event as well as methods and material for treating patients at risk of experiencing a major adverse cardiac event (e.g., patients who underwent percutaneous coronary intervention (PCI) for ST-elevation myocardial infarction (STEMI) are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/873,122, filed on Sep. 3, 2013. The disclosure of the priorapplication is considered part of (and is incorporated by reference in)the disclosure of this application.

BACKGROUND

1. Technical Field

This document relates to methods and materials for reducing the risk ofmajor adverse cardiac events. For example, this document providesmethods and materials for identifying patients at risk of experiencing amajor adverse cardiac event as well as methods and material for treatingpatients at risk of experiencing a major adverse cardiac event (e.g.,patients who underwent percutaneous coronary intervention (PCI) forST-elevation myocardial infarction (STEMI)).

2. Background Information

Strategies to rapidly re-perfuse patients presenting with STEMI haveconsiderably improved acute survivorship. These patients, however,harbor a significant long-term risk of experiencing a major cardiacadverse event following, for example, PCI for STEMI.

SUMMARY

This document provides methods and materials for reducing the risk ofmajor adverse cardiac events. For example, this document providesmethods and materials for identifying patients at risk of experiencing amajor adverse cardiac event as well as methods and material for treatingpatients at risk of experiencing a major adverse cardiac event (e.g.,patients who underwent PCI for STEMI).

As described herein, a STEMI patient who underwent PCI can be assessedto determine whether or not that patient has an increased risk ofexperiencing a major adverse cardiac event as opposed to beingidentified as being unlikely to experience a major adverse cardiacevent. For example, the expression profiles of one or more (e.g., one,two, three, four, five, six, seven, eight, nine, ten, eleven, 12, 13,14, 15, 16, 17, 18, or 19) of the following polypeptides can be used toidentify patients at risk of experiencing a major adverse cardiac event:eotaxin-3, cathepsin-S, Dickopf-1 (DKK-1), follistatin, suppression oftumorigenicity-2 (ST-2), GRO-alpha (GRO-α), interleukin-21 (IL-21),nephroblastoma overexpressed (NOV), transferrin, tissue inhibitor ofmetallopeptidase-2 (TIMP-2), tumor necrosis factor receptor-1 and -2(TNFαRI and II), erythroblastic leukemia viral oncogene-3 (ErBb3),neutrophil-activating protein-2 (NAP-2), angiostatin, chemokineligand-25 (CCL25), angiopoietin like-4 (ANGPTL4), matrixmetalloproteinase-3 (MMP-3), and transforming growth factor-α (TGF-α).In some cases, a myocardial infarction patient or a STEMI patient whounderwent PCI can be treated by administering a NAP-2 polypeptide or anucleic acid encoding a NAP-2 polypeptide to the patient. In some cases,a patient to be treated can be identified for treatment by assessingexpression profiles as described herein. In some cases, the methods andmaterials provided herein can be used to monitor or confirm that aparticular myocardial infarction treatment option (e.g., treatment witha NAP-2 polypeptide or a nucleic acid encoding a NAP-2 polypeptide) iseffective.

In general, one aspect of this document features a method for improvingcardiac function. The method comprises, or consists essentially of,administering a composition comprising a NAP-2 polypeptide or a nucleicacid encoding a NAP-2 polypeptide to a mammal, thereby improving cardiacfunction of said mammal. The composition can comprise the NAP-2polypeptide. The composition can comprise the nucleic acid encoding aNAP-2 polypeptide. The mammal can be a human. The mammal can be a humanpatient who underwent percutaneous coronary intervention forST-elevation myocardial infarction. The method can compriseadministering the composition during a percutaneous coronaryintervention. The method can comprise administering a TGF-α polypeptideor a nucleic acid encoding a TGF-α polypeptide to the mammal.

In another aspect, this document features a method for improving cardiacfunction. The method comprises, or consists essentially of,administering a composition comprising a TGF-α polypeptide or a nucleicacid encoding a TGF-α polypeptide to a mammal, thereby improving cardiacfunction of said mammal. The composition can comprise the TGF-αpolypeptide. The composition can comprise the nucleic acid encoding aTGF-α polypeptide. The mammal can be a human. The mammal can be a humanpatient who underwent percutaneous coronary intervention forST-elevation myocardial infarction. The method can compriseadministering the composition during a percutaneous coronaryintervention.

In another aspect, this document features a method for improving cardiacfunction. The method comprises, or consists essentially of,administering a composition comprising a ErBb3 polypeptide or a nucleicacid encoding a ErBb3 polypeptide to a mammal, thereby improving cardiacfunction of said mammal. The composition can comprise the ErBb3polypeptide. The composition can comprise the nucleic acid encoding aErBb3 polypeptide. The mammal can be a human. The mammal can be a humanpatient who underwent percutaneous coronary intervention forST-elevation myocardial infarction. The method can compriseadministering the composition during a percutaneous coronaryintervention.

In another aspect, this document features a method for improving cardiacfunction. The method comprises, or consists essentially of,administering a composition comprising an inhibitor of eotaxin-3,cathepsin-S, DKK-1, follistatin, ST-2, GRO-α, IL-21, NOV, transferrin,TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4, and/or MMP-3polypeptide expression or activity to a mammal, thereby improvingcardiac function of said mammal. The mammal can be a human. The mammalcan be a human patient who underwent percutaneous coronary interventionfor ST-elevation myocardial infarction. The method can compriseadministering the composition during a percutaneous coronaryintervention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that coronary thrombus aspiration during STEMIprovides the serological resource for high throughput proteomicassessment. (A) is an angiographic depiction of an occlusive thrombus inthe left anterior descending artery. (LAD; left panel) Middle and rightpanels depict the steps taken to aspirate clot and coronary blood out ofthe occluded vessel. (B) Illustration a clot extraction via a coronaryaspiration catheter and collection of sample in a vacuum syringe. Highthroughput proteomic assessment documents the protein content of theoccluded coronary blood at the time of reperfusion in STEMI.

FIG. 2 provides a clinical assessment of protected and vulnerablepatient cohorts. (A) Mayo Clinic Risk Model of major adverse cardiacevents score for each patient at the time of STEMI. (B) The percentageof patients receiving respective pharmacologic agent. (C)Echocardiographic parameters. (D) Ejection fraction change and survivalat two years following STEMI.

FIG. 3 provides cytokine data. (A) Cytokine concentrations in protectedand vulnerable cohorts. (B) ROC curves for each cytokine of FIG. 3A.

FIG. 4 contains bar graphs plotting standard curves for the indicatedcytokine.

FIG. 5 contains bar graphs plotting standard curves for the indicatedcytokine.

FIG. 6 contains bar graphs plotting standard curves for the indicatedcytokine.

FIG. 7 contains bar graphs plotting standard curves for the indicatedcytokine.

FIG. 8 contains bar graphs plotting standard curves for the indicatedcytokine.

FIG. 9 contains bar graphs plotting standard curves for the indicatedcytokine.

FIG. 10 contains bar graphs plotting standard curves for the indicatedcytokine.

FIG. 11 contains bar graphs plotting cytokine concentrations inprotected and vulnerable cohorts.

FIG. 12 contains ROC curves for the indicated cytokines.

FIG. 13 contains a table with cytokine sensitivity and specificity alongwith pathobiological role in myocardial infarction.

FIG. 14 illustrates network analysis. (A) Diagram of an interactionnetwork of 19 identified cytokines (bold font). (B) Network degreedistribution, (P[k]) versus degree (k), and clustering coefficientdistribution, (C[k]) versus (k), indicating non-stochastic scale-freenetwork architecture and network hierarchical tendencies, respectively.(C) Bar graph of prioritized molecular and physiological functions. (D)Bar graph of prioritized canonical pathways.

FIG. 15 demonstrates demonstrate the functional analysis of coronary bedproteins in cardiac ischemia reperfusion injury. (A) A time-line ofanimal experiments: data collection, surgery, and therapy. (B) Data ofPTAH fibrosis staining and quantification in saline (n=20) and growthfactor treated groups (n=10). (C) Data of an echocardiographic analysisof both groups demonstrating functional benefit in growth factor treatedmice (n=6) compared to saline (n=12).

DETAILED DESCRIPTION

This document provides methods and materials for reducing the risk ofmajor adverse cardiac events. For example, this document providesmethods and materials for identifying patients at risk of experiencing amajor adverse cardiac event as well as methods and material for treatingpatients at risk of experiencing a major adverse cardiac event (e.g.,patients who underwent PCI for STEMI). Examples of major adverse cardiacevents include, without limitation, death, heart failure, recurrentmyocardial infarction, and repeat hospitalization for cardiac-relatedevents.

As described herein, the expression levels of one or more (e.g., one,two, three, four, five, six, seven, eight, nine, ten, eleven, 12, 13,14, 15, 16, 17, 18, or 19) of the following polypeptides within a serumsample obtained from a myocardial infarction patient (e.g., a STEMIpatient who underwent PCI) can be used to identify patients at risk ofexperiencing a major adverse cardiac event: eotaxin-3, cathepsin-S,DKK-1, follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2,TNFαRI, TNFαRII, ErBb3, NAP-2, angiostatin, CCL25, ANGPTL4, MMP-3, andTGF-α. For example, if a myocardial infarction patient (e.g., a STEMIpatient who underwent PCI) contains serum (e.g., coronary serum) with anelevated level of one or more of eotaxin-3, cathepsin-S, DKK-1,follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI,TNFαRII, angiostatin, CCL25, ANGPTL4, and MMP-3, then the patient can beclassified as being at risk of experiencing a major adverse cardiacevent. In some cases, if a myocardial infarction patient (e.g., a STEMIpatient who underwent PCI) contains serum (e.g., coronary serum) with areduced level of one or more of ErBb3, NAP-2, and TGF-α, then thepatient can be classified as being at risk of experiencing a majoradverse cardiac event. In some cases, if a myocardial infarction patient(e.g., a STEMI patient who underwent PCI) contains serum (e.g., coronaryserum) with an elevated level of one or more of eotaxin-3, cathepsin-S,DKK-1, follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2,TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4, and MMP-3 and a reducedlevel of one or more of ErBb3, NAP-2, and TGF-α, then the patient can beclassified as being at risk of experiencing a major adverse cardiacevent.

A human eotaxin-3 polypeptide can have the amino acid sequence set forthin GenBank® Accession No. NP_006063.1 (GI No. 10344) and can be encodedby the nucleic acid sequence set forth in GenBank® Accession No.NM_006072 (GI No. 10344). A human cathepsin-S polypeptide can have theamino acid sequence set forth in GenBank® Accession No. NP_004070.3 (GINo. 1520) and can be encoded by the nucleic acid sequence set forth inGenBank® Accession No. NM_004079.4 (GI No. 1520). A human DKK-1polypeptide can have the amino acid sequence set forth in GenBank®Accession No. NP_036374.1 (GI No. 22943) and can be encoded by thenucleic acid sequence set forth in GenBank® Accession No. NM_012242 (GINo. 22943). A human follistatin polypeptide can have the amino acidsequence set forth in GenBank® Accession No. NP_037541.1 (GI No. 10468)and can be encoded by the nucleic acid sequence set forth in GenBank®Accession No. NM_013409.2 (GI No. 10468). A human ST-2 polypeptide canhave the amino acid sequence set forth in GenBank® Accession No.BAA02233 (GI No. 6761) and can be encoded by the nucleic acid sequenceset forth in GenBank® Accession No D12763.1 (GI No 6761). A human GRO-αpolypeptide can have the amino acid sequence set forth in GenBank®Accession No. NP_001502.1 (GI No. 2919) and can be encoded by thenucleic acid sequence set forth in GenBank® Accession No. NM_001511 (GINo. 2919). A human IL-21 polypeptide can have the amino acid sequenceset forth in GenBank® Accession No. NP_068575.1 (GI No. 59067) and canbe encoded by the nucleic acid sequence set forth in GenBank® AccessionNo. NM_021803 (GI No. 59067). A human NOV polypeptide can have the aminoacid sequence set forth in GenBank® Accession No. NP_002505.1 (GI No.4856) and can be encoded by the nucleic acid sequence set forth inGenBank® Accession No. NM_002514 (GI No. 4856). A human transferrinpolypeptide can have the amino acid sequence set forth in GenBank®Accession No. NP_001054.1 (GI No. 7018) and can be encoded by thenucleic acid sequence set forth in GenBank® Accession No. NM_001063.3(GI No. 7018). A human TIMP-2 polypeptide can have the amino acidsequence set forth in GenBank® Accession No. NP_003246.1 (GI No. 7077)and can be encoded by the nucleic acid sequence set forth in GenBank®Accession No. NM_003255.4 (GI No. 7077). A human TNFαRI polypeptide canhave the amino acid sequence set forth in GenBank® Accession No.NP_001056.1 (GI No. 7132) and can be encoded by the nucleic acidsequence set forth in GenBank® Accession No. NM_001065 (GI No. 7132). Ahuman TNFαRII polypeptide can have the amino acid sequence set forth inGenBank® Accession No. NP_001057.1 (GI No. 7133) and can be encoded bythe nucleic acid sequence set forth in GenBank® Accession No. NM_001066(GI No. 7133). A human ErBb3 polypeptide can have the amino acidsequence set forth in GenBank® Accession No. NP_001005915.1 orNP_001973.2 (GI No. 2065) and can be encoded by the nucleic acidsequence set forth in GenBank® Accession No. NM_001005915.1 orNM_001982.3 (GI No. 2065). A human NAP-2 polypeptide can have the aminoacid sequence set forth in GenBank® Accession No. NP_002695.1 (GI No.5473) and can be encoded by the nucleic acid sequence set forth inGenBank® Accession No. NM_002704 (GI No. 5473). A human angiostatinpolypeptide can have the amino acid sequence set forth in GenBank®Accession No. NP_000292 (GI No. 5340) and can be encoded by the nucleicacid sequence set forth in GenBank® Accession No. NM_000301 (GI No.5340). A human CCL25 polypeptide can have the amino acid sequence setforth in GenBank® Accession No. NP_005615.2 (GI No. 6370) and can beencoded by the nucleic acid sequence set forth in GenBank® Accession No.NM_005624 (GI No. 6370). A human ANGPTL4 polypeptide can have the aminoacid sequence set forth in GenBank® Accession No. NP_001034756.1 orNP_647475.1 (GI No. 51129) and can be encoded by the nucleic acidsequence set forth in GenBank® Accession No. NM_001039667.1 orNM_139314.1 (GI No. 51129). A human MMP-3 polypeptide can have the aminoacid sequence set forth in GenBank® Accession No. NP_002413.1 (GI No.4314) and can be encoded by the nucleic acid sequence set forth inGenBank® Accession No. NM_002422 (GI No. 4314). A human TGF-αpolypeptide can have the amino acid sequence set forth in GenBank®Accession No. NP_003227.1 (GI No. 7039) and can be encoded by thenucleic acid sequence set forth in GenBank® Accession No. NM_003236 (GINo. 7039).

The term “elevated level” as used herein with respect to the level of apolypeptide (e.g., an eotaxin-3, cathepsin-S, DKK-1, follistatin, ST-2,GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI, TNFαRII, angiostatin,CCL25, ANGPTL4, or MMP-3 polypeptide) is any level that is greater than(e.g., at least about 10, 15, 20, or 25 percent greater than) areference level for that polypeptide. The term “reduced level” as usedherein with respect to the level of a polypeptide (e.g., an ErBb3,NAP-2, or TGF-α polypeptide) is any level that is less than (e.g., atleast about 10, 15, 20, or 25 percent less than) a reference level forthat polypeptide. The term “reference level” as used herein with respectto an eotaxin-3, cathepsin-S, DKK-1, follistatin, ST-2, GRO-α, IL-21,NOV, transferrin, TIMP-2, TNFαRI, TNFαRII, ErBb3, NAP-2, angiostatin,CCL25, ANGPTL4, MMP-3, or TGF-α polypeptide is the level of expressionof that polypeptide typically observed by healthy humans or humanpatients with a low risk of experiencing a major adverse cardiac event.For example, a reference level of eotaxin-3 expression can be theaverage level of eotaxin-3 expression that is present in samplesobtained from a random sampling of 50 healthy humans without evidence ofcardiac problems. It will be appreciated that levels from comparablesamples are used when determining whether or not a particular level isan elevated level or a reduced level. In some cases, the reference levelof polypeptide expression can be a ratio of an expression value of thatpolypeptide in a sample to an expression value of a control polypeptidein the sample. A control polypeptide can be any polypeptide that has aminimal variation in expression level across various samples of the typefor which the polypeptide serves as a control. For example, albuminpolypeptides, C-reactive protein, or NT-proBNP polypeptides can be usedas control polypeptides. In some cases, the reference level ofpolypeptide expression can be a ratio of an expression value of thatpolypeptide in a sample to the level of total protein in the sample.

An elevated level of eotaxin-3, cathepsin-S, DKK-1, follistatin, ST-2,GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI, TNFαRII, angiostatin,CCL25, ANGPTL4, or MMP-3 polypeptide expression can be any levelprovided that the level is at least about 10 percent greater than (e.g.,at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 percentgreater than) a corresponding reference level. For example, an elevatedlevel of eotaxin-3 expression can be 15 or more percent greater than thereference level for eotaxin-3 expression.

A reduced level of ErBb3, NAP-2, or TGF-α polypeptide expression can beany level provided that the level is at least about 10 percent less than(e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 percentless than) a corresponding reference level. For example, a reduced levelof ErBb3 expression can be 15 or more percent less than the referencelevel for ErBb3 expression.

Any appropriate method can be used to determine expression levels of apolypeptide within a serum sample. For example, ELISA and otherimmunological-based assays can be used to determine the level ofeotaxin-3, cathepsin-S, DKK-1, follistatin, ST-2, GRO-α, IL-21, NOV,transferrin, TIMP-2, TNFαRI, TNFαRII, ErBb3, NAP-2, angiostatin, CCL25,ANGPTL4, MMP-3, and/or TGF-α within a serum sample.

Once the levels of one or more of eotaxin-3, cathepsin-S, DKK-1,follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI,TNFαRII, ErBb3, NAP-2, angiostatin, CCL25, ANGPTL4, MMP-3, and/or TGF-αpolypeptide expression in a sample from a patient is determined, thenthe levels can be compared to reference levels and used to evaluate thelikelihood that the patient will experience a major adverse cardiacevent. Those patients determined to be likely to experience a majoradverse cardiac event as described herein can be subjected to increasedmonitoring and/or can be treated with an appropriate treatment option.For example, a patient identified as being likely to experience a majoradverse cardiac event as described herein can be treated with aggressivepharmacotherapy (e.g., beta-adrenoceptor blockade treatments, treatmentwith angiotensin converting enzyme inhibitors, aldosterone antagonismtreatments, and/or treatment with antiplatelet agents), hemodynamicsupport (e.g., intra-aortic balloon pump and/or mechanical augmentationof cardiac output), surgical intervention (e.g., coronary bypassgrafting or left ventricular assist device placement), and/ordevice-based intervention (e.g., resychronization therapy or implantablecardiac defibrillators).

In some cases, a patient at risk of experiencing a major adverse cardiacevent (e.g., a patient identified as being likely to experience a majoradverse cardiac event as described herein) can be treated by increasingthe level of NAP-2 polypeptide expression, by increasing the level ofTGF-α polypeptide expression, by increasing the level of ErBb3polypeptide expression, or by increasing the levels of a combination ofany two of NAP-2 polypeptide expression, TGF-α polypeptide expression,and ErBb3 polypeptide expression (e.g. a combination of both NAP-2polypeptide and TGF-α polypeptide expression). In some cases, a patientat risk of experiencing a major adverse cardiac event (e.g., a patientidentified as being likely to experience a major adverse cardiac eventas described herein) can be treated by increasing the level of NAP-2polypeptide expression, by increasing the level of TGF-α polypeptideexpression, and by increasing the level of ErBb3 polypeptide expression.An increased level of NAP-2 polypeptide, TGF-α polypeptide expression,and/or ErBb3 polypeptide expression can be used to reduce scar size andtissue remodeling and to improve cardiac function. For example, an areaof fibrosis reflecting scar size from injury can be reduced by 10 to 100percent (e.g., by 10 to 90 percent, by 10 to 80 percent, by 10 to 70percent, by 10 to 60 percent, by 10 to 50 percent, by 20 to 100 percent,by 30 to 100 percent, or by 20 to 60 percent) following administrationof NAP-2 polypeptides or nucleic acid encoding a NAP-2 polypeptide,TGF-α polypeptides or nucleic acid encoding a TGF-α polypeptide, ErBb3polypeptides or nucleic acid encoding a ErBb3 polypeptide, orcombinations thereof. In some cases, cardiac tissue remodeling can bereduced by 10 to 100 percent (e.g., by 10 to 90 percent, by 10 to 80percent, by 10 to 70 percent, by 10 to 60 percent, by 10 to 50 percent,by 20 to 100 percent, by 30 to 100 percent, or by 20 to 60 percent)following administration of NAP-2 polypeptides or nucleic acid encodinga NAP-2 polypeptide, TGF-α polypeptides or nucleic acid encoding a TGF-αpolypeptide, ErBb3 polypeptides or nucleic acid encoding a ErBb3polypeptide, or combinations thereof. Examples of improved cardiacfunction include, without limitation, increased survivorship, reducedhospitalization, symptom-free tolerance of physical activity, improvedglobal physical fitness, improved cardiac ejection fraction, improvedcardiac output, improved stroke volume, improved cardiac mass index, andreduced scar size.

In some cases, the level of NAP-2 polypeptide, TGF-α polypeptide, and/orErBb3 polypeptide expression can be increased by administering acomposition containing NAP-2, TGF-α, and/or ErBb3 polypeptides. In somecases, the level of NAP-2 polypeptide, TGF-α polypeptide, and/or ErBb3polypeptide expression can be increased by administering one or morenucleic acids (e.g., DNA or RNA) encoding a NAP-2 polypeptide, TGF-αpolypeptide, and/or ErBb3 polypeptide to cells of the patient (e.g.,resident or exogenously grown stem cells). Such a nucleic acid canencode a full-length NAP-2 polypeptide, a full-length TGF-α polypeptide,and/or a full-length ErBb3 polypeptide. In some cases, a nucleic acidencoding a fragment of a NAP-2 polypeptide, a TGF-α polypeptide, and/oran ErBb3 polypeptide that retains at least some biological activity canbe used as described herein to reduce scar size and tissue remodelingand/or to improve cardiac function.

A nucleic acid encoding a NAP-2 polypeptide, a TGF-α polypeptide, and/oran ErBb3 polypeptide (or a fragment thereof) can be administered to apatient using any appropriate method. For example, a nucleic acid can beadministered to a human using a vector such as a viral vector.

Vectors for administering nucleic acids (e.g., a nucleic acid encoding aNAP-2 polypeptide, a TGF-α polypeptide, and/or an ErBb3 polypeptide (orfragments thereof)) to a mammal are known in the art and can be preparedusing standard materials (e.g., packaging cell lines, helper viruses,and vector constructs). See, for example, Gene Therapy Protocols(Methods in Molecular Medicine), edited by Jeffrey R. Morgan, HumanaPress, Totowa, N.J. (2002) and Viral Vectors for Gene Therapy: Methodsand Protocols, edited by Curtis A. Machida, Humana Press, Totowa, N.J.(2003). Virus-based nucleic acid delivery vectors are typically derivedfrom animal viruses, such as adenoviruses, adeno-associated viruses,retroviruses, lentiviruses, vaccinia viruses, herpes viruses, andpapilloma viruses. Lentiviruses are a genus of retroviruses that can beused to infect cells. Adenoviruses contain a linear double-stranded DNAgenome that can be engineered to inactivate the ability of the virus toreplicate in the normal lytic life cycle. Adenoviruses andadeno-associated viruses can be used to infect cells.

Vectors for nucleic acid delivery can be genetically modified such thatthe pathogenicity of the virus is altered or removed. The genome of avirus can be modified to increase infectivity and/or to accommodatepackaging of a nucleic acid, such as a nucleic acid encoding a NAP-2polypeptide, a TGF-α polypeptide, and/or an ErBb3 polypeptide (or afragment thereof). A viral vector can be replication-competent orreplication-defective, and can contain fewer viral genes than acorresponding wild-type virus or no viral genes at all.

In addition to nucleic acid encoding a NAP-2 polypeptide, a TGF-αpolypeptide, and/or an ErBb3 polypeptide (or a fragment thereof), aviral vector can contain regulatory elements operably linked to anucleic acid encoding the polypeptide(s) (or a fragment thereof). Suchregulatory elements can include promoter sequences, enhancer sequences,response elements, signal peptides, internal ribosome entry sequences,polyadenylation signals, terminators, or inducible elements thatmodulate expression (e.g., transcription or translation) of a nucleicacid. The choice of element(s) that may be included in a viral vectordepends on several factors, including, without limitation, inducibility,targeting, and the level of expression desired. For example, a promotercan be included in a viral vector to facilitate transcription of anucleic acid encoding a NAP-2 polypeptide, a TGF-α polypeptide, and/oran ErBb3 polypeptide. A promoter can be constitutive or inducible (e.g.,in the presence of tetracycline), and can affect the expression of anucleic acid encoding a NAP-2 polypeptide, a TGF-α polypeptide, and/oran ErBb3 polypeptide in a general or tissue-specific manner.Tissue-specific promoters include, without limitation, acardiac-specific MHC promoter, a troponin promoter, and an MLC2vpromoter.

As used herein, “operably linked” refers to positioning of a regulatoryelement in a vector relative to a nucleic acid in such a way as topermit or facilitate expression of the encoded polypeptide. For example,a viral vector can contain a cardiac-specific promoter and a nucleicacid encoding a NAP-2 polypeptide, a TGF-α polypeptide, and/or an ErBb3polypeptide. In this case, a cardiac-specific MHC promoter is operablylinked to a nucleic acid encoding a NAP-2 polypeptide, a TGF-αpolypeptide, and/or an ErBb3 polypeptide such that it drivestranscription in cardiac cells.

In some cases, a nucleic acid encoding a NAP-2 polypeptide, a TGF-αpolypeptide, and/or an ErBb3 polypeptide (or a fragment thereof) can beadministered to cells using non-viral vectors. Methods of usingnon-viral vectors for nucleic acid delivery are known to those ofordinary skill in the art. See, for example, Gene Therapy Protocols(Methods in Molecular Medicine), edited by Jeffrey R. Morgan, HumanaPress, Totowa, N.J. (2002). For example, a nucleic acid encoding a NAP-2polypeptide, a TGF-α polypeptide, and/or an ErBb3 polypeptide can beadministered to a mammal by direct injection of nucleic acid molecules(e.g., plasmids) comprising nucleic acid encoding a NAP-2 polypeptide, aTGF-α polypeptide, and/or an ErBb3 polypeptide, or by administeringnucleic acid molecules complexed with lipids, polymers, or nanospheres.

A nucleic acid encoding a NAP-2 polypeptide, a TGF-α polypeptide, and/oran ErBb3 polypeptide (or a fragment thereof) can be produced by standardtechniques, including, without limitation, common molecular cloning,polymerase chain reaction (PCR), chemical nucleic acid synthesistechniques, and combinations of such techniques. For example PCR orRT-PCR can be used with oligonucleotide primers designed to amplifynucleic acid (e.g., genomic DNA or RNA) encoding a NAP-2 polypeptide, aTGF-α polypeptide, and/or an ErBb3 polypeptide (or a fragment thereof).

In some cases, a patient at risk of experiencing a major adverse cardiacevent (e.g., a patient identified as being likely to experience a majoradverse cardiac event as described herein) can be treated by reducingthe level of expression of one or more (e.g., one, two, three, four,five, six, seven, eight, nine, ten, or more) of the followingpolypeptides: eotaxin-3, cathepsin-S, DKK-1, follistatin, ST-2, GRO-α,IL-21, NOV, transferrin, TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25,ANGPTL4, and/or MMP-3 polypeptides. A reduction in the level of one ormore of these polypeptides can be used to reduce scar size and tissueremodeling and to improve cardiac function. For example, an area offibrosis reflecting scar size from injury can be reduced by 10 to 100percent (e.g., by 10 to 90 percent, by 10 to 80 percent, by 10 to 70percent, by 10 to 60 percent, by 10 to 50 percent, by 20 to 100 percent,by 30 to 100 percent, or by 20 to 60 percent) following administrationof a composition designed to reduce eotaxin-3, cathepsin-S, DKK-1,follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI,TNFαRII, angiostatin, CCL25, ANGPTL4, and/or MMP-3 polypeptideexpression or activity. In some cases, cardiac tissue remodeling can bereduced by 10 to 100 percent (e.g., by 10 to 90 percent, by 10 to 80percent, by 10 to 70 percent, by 10 to 60 percent, by 10 to 50 percent,by 20 to 100 percent, by 30 to 100 percent, or by 20 to 60 percent)following administration of a composition designed to reduce eotaxin-3,cathepsin-S, DKK-1, follistatin, ST-2, GRO-α, IL-21, NOV, transferrin,TIMP-2, TNFαRI, TNFαRII, angiostatin, CCL25, ANGPTL4, and/or MMP-3polypeptide expression or activity. Examples of improved cardiacfunction include, without limitation, increased survivorship, reducedhospitalization, symptom-free tolerance of physical activity, improvedglobal physical fitness, improved cardiac ejection fraction, improvedcardiac output, improved stroke volume, improved cardiac mass index, andreduced scar size.

In some cases, the level of eotaxin-3, cathepsin-S, DKK-1, follistatin,ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI, TNFαRII,angiostatin, CCL25, ANGPTL4, and/or MMP-3 polypeptide expression can bereduced by administering a composition containing an antisense or RNAimolecule (e.g., an siRNA molecule or an shRNA molecule) designed toreduce polypeptide expression of an eotaxin-3, cathepsin-S, DKK-1,follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI,TNFαRII, angiostatin, CCL25, ANGPTL4, or MMP-3 polypeptide. In somecases, the level of eotaxin-3, cathepsin-S, DKK-1, follistatin, ST-2,GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI, TNFαRII, angiostatin,CCL25, ANGPTL4, or MMP-3 polypeptide activity can be reduced byadministering an inhibitor of eotaxin-3, cathepsin-S, DKK-1,follistatin, ST-2, GRO-α, IL-21, NOV, transferrin, TIMP-2, TNFαRI,TNFαRII, angiostatin, CCL25, ANGPTL4, or MMP-3 polypeptide activity.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1—Differential Coronary Serum Proteome Signature inHumans Suffering from Acute Myocardial Infarction Identifies VulnerablePatients

Study Design

Patients between 40 and 82 years of age underwent coronary thrombusaspirate prior to PCI for STEMI. Fresh coronary aspirates were stored inEDTA tubes and centrifuged at 4° C. Serum supernatant was collected,treated with protease inhibitor, split into working aliquots, flashfrozen in liquid nitrogen, and stored at −80° C. within 60 minutes ofcoronary sampling. The Mayo Clinic Risk Model of MACE and TIMI scoringwere utilized for stratification at the time of myocardial infarction,and patients identified as non-high risk were enrolled and followed(n=25). Those suffering from death, recurrent infarction, or heartfailure (MACE) within a two-year follow-up were categorized asvulnerable (n=11), while those who did not were categorized as protected(n=14).

Proteomics Evaluation

Stored coronary serum samples were thawed only once and prepared forELISA-based antibody array analysis (Quantibody Human Array-6000,RayBiotech) to quantify 280 distinct cytokines. Serum samples werediluted 1:5 with sample diluent. Glass protein array slides were allowedto equilibrate and dry at room temperature for 45 minutes. Each chamberwithin the slide was blocked for 30 minutes. Standards for each cytokinewere prepared as eight serial dilutions and added to respectivechambers, while the remaining chambers received 100 μL of serum.Following overnight incubation at 4° C., slides were washed for a totalof 35 minutes. A biotinylated labeling agent was added to each chamberand incubated overnight at 4° C. Slides were washed for an additional 35minutes and incubated with Cy3-Streptavidin for 1 hour protected fromlight at room temperature. Slides were washed and centrifuged for 3minutes at 1,000 rpm. Dry slides were analyzed using Molecular DevicesAxon GenePix Pro 6 software to generate a standard curve for eachcytokine and determine individual concentrations in coronary serum.

Network Analysis

Differentially expressed array proteins were submitted for networkanalysis using Ingenuity Pathways Knowledge Base (IPKB, IngenuitySystems, “www” dot “ingenuity.com”) to identify associated functionalsub-networks. These were merged into a composite interactome in IPKB anddepicted using the network visualization program Cytoscape 2.8.2, withnetwork topology characterized using Network Analyzer (Smoot et al.,Bioinformatics, 27(3):431-2 (2011)). Computed properties included nodedegree (k), degree distribution (P[k]), and clustering coefficientdistribution (C[k]), the derivation of which were described elsewhere(Arrell et al., Stem Cells, 26(2):387-400 (2008)); Zlatkovic-Lindor etal., Stem Cells, 28(8):1355-67 (2010); and Crespo-Diaz et al., CellTransplant., 20(6):797-811 (2011)). IPKB also prioritizedover-represented molecular and physiological functions and canonicalpathways associated with the resolved interaction network.

Animal Studies

Surgery was performed on 48 mice age 8- to 12-week old C57BL/6 under1-2% isoflurane. Left anterior descending artery (LAD) was temporarilyligated for 60 minutes with the animal anesthetized throughout this timeperiod. This was followed by restoration of blood flow for 5-10 minutes.Region supplied by LAD was then injected 5 times with 2.5 μL of salineor growth factors. The concentrations used for NAP-2 and TGF-αinjections were 5-25 ng per injection and 4-20 ng per injection,respectively.

Pain prophylaxis was implemented by an acetaminophen regimen (100-300mg/kg in drinking water) for 2 days prior to and 5 days after surgery.Prior to surgery, mice were randomized into saline (n=12) or growthfactor treated (n=6) groups in 2:1 format. Individuals involved inperforming the surgery and collecting and analyzing echocardiographicdata were blinded throughout the study. Cardiac function and structurewere quantified prospectively by echocardiography using a 30 MHztransducer up to 1 month following ischemia reperfusion injury. Ejectionfraction was defined as [(LVVd−LVVs)/LVVd]×100, where LVVd is LVend-diastolic volume, and LVVs is LV end-systolic volume.

Statistical Analysis

This work was designed to assess the serological changes in theconcentration of 280 cytokines. Patient clinical data were analyzed asmean±SD and compared between groups with 2-sample student t-test ormedian±interquartile range. Cytokine concentrations were presented asmedian±interquartile range, and analyzed by non-parametric statisticsMann-Whitney U test. Differences were considered significant withp<0.05. Receiver operating characteristic (ROC) curves were constructedto evaluate the prognostic potential of each cytokine for STEMI patientstratification prior to PCI. Network analysis p-values were calculatedusing Fisher's exact test, determining the probability that associationbetween dataset proteins and functions or canonical pathways isexplained by chance alone. Statistical analyses were performed in SASJMP 9.0 and MedCalc software, version 12.2.1.

Results

Coronary Serum Molecular Fingerprinting

Coronary thrombus aspirate was obtained from STEMI patients with anocclusive coronary lesion. Thrombectomy was performed prior to PCI witha drug-eluting stent (FIG. 1A). Patient aspirates were included in thestudy if coronary intervention was without complication and restoredfrom TIMI-0 to TIMI-3 flow. All patients were managed according toACC/AHA practice guidelines (Kushner et al., J. Am. Coll. Cardiol.,54(23):2205-41 (2009)). Aspirated thrombus and coronary blood wasprocessed for plasma extraction and subjected to proteomic assessmentwith generation of a heat map plotting the plasma protein expressionprofile for each patient (FIG. 1B).

Discrimination of Vulnerable Versus Protected STEMI Cohorts

During a 2-year follow-up period, patients with major cardiac adverseevents were categorized as vulnerable (n=11), and those without werecategorized as protected (n=14). No differences in demographics andcardiovascular health factors (Table 1) were noted between the twocohorts. Several risk stratification models were calculated for eachpatient. Specifically, TIMI risk score for probability of death duringhospitalization and up to 6-months was low in both patient cohorts(5.5±2 in vulnerable and 4±4.5 in protected, p=0.08) (Table 1) (Morrowet al., JAMA, 286(11):1356-9 (2001) and Morrow et al., Circulation,102(17):2031-7 (2000)). The Mayo Clinic Risk Model of MACE validatedTIMI scoring, placing both groups in the non-high risk category (5.5±3in vulnerable and 4±2 in protected, p=0.22) (FIG. 2A) (Singh et al., J.Am. Coll. Cardiol., 40(3):387-93 (2002)). Echocardiographic evaluation,however, revealed severe reduction in ejection fraction (−18±7%) in thevulnerable group compared to limited change (−0.5±2.2%) in the protectedgroup (FIG. 2D; p<0.05). Survivorship was 65% in the vulnerable versus100% in the protected group (FIG. 2D).

TABLE 1 Baseline characteristics of STEMI patient group. Protectedcohort Vulnerable cohort Baseline Characteristics n = 14 n = 11 Age inyears 67 (8) 69.5 (10) Women, % 50% 40% Troponin levels, median ng/ml 4(3.22) 8 (16.3) C-reactive protein, mg/liter 3 (11.4) 3 (12.3) TIMI RiskIndex 3 (3.5) 5.5 (3.5) WBC levels 11620 (3790) 12795 (8223) MIpresentation to Balloon, hrs 6 hrs 5 hrs NYHA Score, median 1 (0.5) 1(0.75) Past family history CAD 5 2 Hypercholesterolemia 4 4 Hypertension5 2 Smoking 4 1 Diabetes Mellitus 1 2Proteomics Dissection Uncovers Vulnerability Biomarkers

Coronary serum aspirates from each patient were evaluated for proteincontent. Standard curves for each of the 280 probes, constituting acomprehensive cytokine panel, were generated to determine concentration.Initially, a p≤0.075 cutoff was utilized to capture a spectrum ofcandidate cytokines with differential concentrations in protected versusvulnerable patient samples. These included eotaxin-3, cathepsin-S,Dickopf-1 (DKK-1), follistatin, suppression of tumorigenicity-2 (ST-2),GRO-alpha (GRO-α), interleukin-21 (IL-21), nephroblastoma overexpressed(NOV), transferrin, tissue inhibitor of metallopeptidase-2 (TIMP-2),tumor necrosis factor receptor-1 and -2 (TNFαRI and II), erythroblasticleukemia viral oncogene-3 (ErBb3), neutrophil-activating protein-2(NAP-2), angiostatin, chemokine ligand-25 (CCL25), angiopoietin like-4(ANGPTL4), matrix metalloproteinase-3 (MMP-3) and transforming growthfactor-α (TGF-α) (FIGS. 3A and 4-10).

The resulting 19 cytokines were subjected to a more discriminatingcutoff (p≤0.025) yielding six cytokines, namely NAP-2, angiostatin,CCL25, ANGPTL4, MMP-3, and TGF-α. The median concentrations for each ofthe six resolved factors in the protected and vulnerable group were for:NAP-2 8.0 ng/mL and 5.0 ng/mL (p=0.005), Angiostatin 146 pg/mL and 242pg/mL (p=0.01), CCL25 undetected and 3.8 ng/mL (p=0.025), ANGPTL4 73.3pg/mL and 152 pg/mL (p=0.019), MMP-3 884 pg/mL and 2.7 ng/mL (p=0.014),and TGF-α 2.1 ng/mL and undetected (p=0.004), respectively (FIG. 3A). Toprobe the discriminatory potential of each factor, areceiver-operating-characteristic (ROC) curve was generated (FIG. 3B)yielding area under the curve (AUC) as follows: TGF-α 0.834 (p=0.0003),NAP-2 0.821 (p=0.0005), Angiostatin 0.805 (p=0.001), MMP-3 0.792(p=0.003), ANGPTL4 0.779 (p=0.006) and CCL25 0.759 (p=0.009). This inturn allowed projection of sensitivity and specificity (Table 2).Concentration results (FIG. 11), ROC curves (FIG. 12), and sensitivityand specificity results (FIG. 13) for the remaining 13 factors(0.03≤p≤0.075) were obtained.

TABLE 2 Cytokines with projected sensitivity and specificity; andpathobiological role in myocardial infarction. Sensitivity SpecificityBiomarker (%) (%) Biological role in MI NAP-2 80 90 Activates leukocyteand endothelial cells within atherosclerotic plaque. Angiostetin 100 55Inhibits col ateral coronary vasculature formation. CCL-25 85 64Attracts macrophages and other inflammatory cel s towardsatherosclerotic plaque. ANGPTL-4 80 82 Potentially inhibitsneovascularization. MMP-3 80 73 Extracellular matrix remodeling,associated with left ventricular dysfunction. TGFα 93 73 Used tostimulate stem cell secretion of VEGF to improve myocardial functionpost-MI.Systems Biology Maps Over-represented Processes in MACE ProneIndividuals

The biological relationship between all discriminatory polypeptides wasprobed using complex network analysis. These 19 factors clustered intoan organized network composed of 65 nodes linked by 417 pairwiseconnections (FIG. 14A). Network topology displayed non-stochasticarchitecture with hierarchical tendencies (FIG. 14B). Evaluation ofover-represented molecular and physiological functions revealedprioritization for hematological, immunological, and cardiovascularfunctions (FIG. 14C). Canonical pathway assessment ranked calciumregulation, retinoic acid signaling, and endothelial inflammation as themost highly correlated to the resolved network (FIG. 14D). Thus,derivation of the non-stochastic network encompassing 19 factorsidentified within the coronary serum, maps activated pathophysiologicalprocesses discriminating vulnerable from protected patients at the timeof STEMI.

Therapeutic Effect of Coronary Serum Protein in Ischemia ReperfusionInjury

The day before surgery 48 baseline echocardiographic recordings werecollected, and the mice were randomized 2:1 into saline and growthfactor treated groups. After ischemia reperfusion injury was induced,5-25 ng of NAP-2 and 4-20 ng TGF-α were injected in the region suppliedby LAD (FIG. 15A). Three days later, hearts we harvested, and fibrosiswas quantified on saline (n=20) and growth factor (n=10) treated groups.Hearts treated with growth factors exhibited 14.3±6.2% of fibrosis inthe left ventricular wall compared to 33.8±8.37% in the saline group(p=<0.01) (FIG. 15B).

Echocardiography collection was performed 1 and 10 days, and 4 weeksfollowing injury (FIG. 15A). Significant improvement was observed duringthe acute phases of injury (FIG. 15C) in growth factor treated mice.Left ventricular end-diastolic and systolic volumes were significantlyimproved in the growth factor treated cohort, demonstrating reducedremodeling and organ decompensation. These results demonstrate thatfactors within the coronary bed can be used as molecular therapy toreduce scar size, limit tissue remodeling and improve cardiac functionfollowing STEMI.

Taken together, these results demonstrate that markers such as the 19cytokines described herein can be used to assess long-term outcomesfollowing infarction. High-throughput proteomics thus can provide amolecular snapshot of disease entities at the tissue level. Theseresults also demonstrate that treatment with NAP-2, TGF-α, or bothduring ischemia reperfusion injury can be used to reduce scar size andtissue remodeling, and to improve cardiac function. In addition,real-time monitoring of patient response to injury and/or treatment canbe performed to inform personalized management at the time ofreperfusion or during various treatment or post-treatment phases.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for modulating immune system activity inheart tissue following cardiac injury, the method comprising:administering a composition comprising a neutrophil-activating protein-2(NAP-2) polypeptide to heart tissue of a mammal, thereby modulatingimmune system activity in the heart tissue.
 2. The method of claim 1,wherein said mammal is a human.
 3. The method of claim 1, wherein saidmethod comprises administering said composition during a percutaneouscoronary intervention.
 4. The method of claim 1, wherein the cardiacinjury comprises ischemic reperfusion injury.